Detecting and differentiating neurotransmitters using ultraviolet plasmonic engineered native fluorescence

Detecting neurotransmitters with high sensitivity and selectivity is important to understand their roles in biological functions. Current detection methods for neurotransmitters suffer from poor sensitivity or selectivity. In this article, we propose ultraviolet (UV) plasmonic engineered native fluorescence as a new sensing mechanism to detect neurotransmitters with high sensitivity and selectivity. We measured the native fluorescence of three monoamine neurotransmitters, dopamine (DA), norepinephrine (NE), and 3,4-dihydroxyphenylacetic acid (DOPAC). The average net enhancement and total photon yield enhancement on an aluminum hole array with 300 nm hole spacing substrate were found to be 50× and 60×, for the three molecules. We also observed a 1.5–1.7× reduction in the dominant photon bleaching rate on an aluminum hole array compared to an aluminum-thin film substrate. The photobleaching rates of the native fluorescence of DA, NE and DOPAC were found to be highly sensitive to their molecular structures and can be further engineered by UV plasmonic substrates. The differences in the photobleaching rates for DA and NE were 2× and 1.6× larger on an aluminum thin film and an aluminum hole array than on a silicon substrate. As a proof-of-concept experiment, we mixed DA with NE at different concentration ratios and measured the average photobleaching rates of the mixture. We found that the average photobleaching rate is proportional to the concentration of NE in the mixture. Our findings demonstrate the potential of UV plasmonic engineered native fluorescence to achieve sensitive and selective detection of neurotransmitters.


Fig. S3
The integrated fluorescence intensity S(t) versus exposure time for DA deposited on (a) silicon, (b) an Al 30nm thin film and (c) a P300, NE on (d) silicon, (e) an Al 30nm thin film and (f) a P300, and DOPAC on (g) silicon, (h) an Al 30nm thin film and (i) a P300.The solid red line is the two exponential curve fitting.

Table S1
The fitting decay rate k1 and k2 of the integrated fluorescence intensity S(t) of DA, NE and DOPAC deposited on silicon, Al 30nm thin film and P300.The decay rates are the two exponential curve fitting parameters fitted on every five different spots on the sample (() =  × exp( , ) +  × exp( / )).From Table S1, we calculated that the differences in k1 between DA and NE are larger than 3 times the standard deviations (σ) of DA or NE's k1 (∆k1 > 3σ) on silicon and aluminum hole array.The differences in k1 between DA and NE are larger than 2 times the standard deviations (∆k1 > 2σ) on aluminum thin film.The differences in k1 between NE and DOPAC are larger than 3 times the standard deviations (∆k1 > 3σ) for all 3 substrates.The differences in k1 between DA and DOPAC are larger than 3 times the standard deviations (∆k1 > 3σ) for silicon, and 2 times the standard deviations (∆k1 > 2σ) for aluminum thin film or aluminum hole array.We believe the differences in k1 between neurotransmitters are significant.

Silicon
Table S2 The fitting amplitude a and b of the integrated fluorescence intensity S(t) of DA, NE and DOPAC deposited on silicon, Al 30nm thin film and P300.The decay rates are the two exponential curve fitting parameters fitted on every five different spots on the sample (() =  × exp( , ) +  × exp( / )).The quantum yield refers to the proportion of photons that are emitted compared to the number of photons that are absorbed 1 .The most common way of determining the quantum yield (Φ) of samples is the "Relative Method," which requires knowing the absorbance of both the reference and the sample solution and relies on using well-characterized reference standards with known ΦR value and optical properties closely matching the sample of interest 1,2 .It compares the integrated fluorescence intensity of a sample of known ΦR, generally referred to as the reference, against the samples with unknown ΦS.This method is only applicable to samples that can go into solution because the measurement requires knowledge of the refractive index of the solvent and the absorbance of both reference and sample 1 .It uses a conventional fluorescence spectrometer which detects only a fraction of the light emitted due to a wide range of factors, including the refractive index of the solvent, the scattering of light by the sample, the emission wavelength, the 90° arrangement of the excitation and emission optics.

Silicon
The standard samples should be chosen to ensure they absorb at the excitation wavelength of choice for the test sample and, if possible, emit in a similar region to the test sample.The standard sample must be well-characterized and suitable for such use.The quantum yields of the known standard compounds are primarily independent of excitation wavelength 1 .Therefore, we used aqueous Tryptophan as a standard solution to measure the quantum yield (ΦS) of DA, NE, and DOPAC dissolved in water at concentrations of 0, 10, 20, 30, 40, 50 micromolar using the following formula: where Φ is the quantum yield, I is the integrated intensity, OD is the optical density, and n is the refractive index.The subscript R refers to the reference fluorophore of known quantum yield.Optical density (OD) is related to the speed of light through the medium and takes refraction into account, but absorbance (A) does not take the refraction of light into account and only considers the amount of light lost.We can ignore the differences between OD and A since the refraction of light is negligible.The following equation can be used to calculate A or OD from transmittance (T).
The ratio of integrated intensity per Optical Density can be obtained by the plot of integrated fluorescence intensity versus absorbance.In order to do so, Firstly, the absorbance at the excitation wavelength of samples in 6 different concentrations was recorded using a UV-vis spectrometer.Then, the fluorescence intensity of samples was also recorded using Fluorometer.After calculating the integrated fluorescence intensity, a graph of integrated fluorescence intensity versus absorbance can be plotted.The fluorescence intensity of samples and the standard were measured with identical spectrometer settings such as excitation wavelength, slit widths of excitation and emission monochromator, scan speed, and integration time 3 .The linear regression of data points gives a straight line with the slope of m; this gradient is equal to the ratio of I/OD; therefore, equation ( 1) can be rewritten like below: Having used dilute concentrations in micromolar ranges of DA, DOPAC, and NE dissolved in DI water and also choosing Tryptophan in DI water as the reference standard, there would be no significant change in refractive indexes because the solvent used in the samples and the standard solution is the same.Fig. 2c in the main article, plots the integrated fluorescence intensity of DA, DOPAC and NE for each concentration and their absorbance at the excitation wavelength.The reason for choosing the range of 0 to 50 µM concentration for the compounds was to minimize reabsorption effects 4 .Absorbances in the 10 mm fluorescence cuvette should never exceed 0.1 at and above the excitation wavelength.Above this level, non-linear may be observed due to inner filter effects, and the resulting quantum yield values may be perturbed.
Using the quantum yield value of 13% for Tryptophan in water 1 , one of the good fluorescent standard solutions, available in literatures, the quantum yield of the three tested substances including DA, DOPAC, and NE was reported in Table S3.Propagation of error is used to estimate the uncertainty in quantum yield calculation for each component.
The propagated uncertainty associated with the quantum yield values (±0.0080 for DA, ±0.0021 for DOPAC and ±0.0062 for NE) represent the standard errors 5 in the measurements and reflect the precision of the experimental data.